The present invention relates to a method and an agent for the photometric detection and assay of hydroperoxides per se, or indirectly, of substances which react to liberate hydroperoxides, in liquids containing proteins.
The determination of hydroperoxides, in particular hydrogen peroxide, plays an important part in clinical chemistry. Many enzymatic substrate determinations are carried out with oxidases. For example, cholesterol is assayed with cholesterol oxidase; glucose is assayed with glucose oxidase; aminoacids are assayed with aminoacid oxidase; uric acid is assayed with uricase and the like. The hydrogen peroxide formed in amounts equimolar with those of the substance being assayed in these reactions, can be determined in a known manner electrochemically or photometrically. The electrochemical processes are disadvantageous because they require special apparatus and tend to be unreliable. Photometric methods of determination are preferred and more widely used.
One of the most widely known photometric methods of determination is based on the oxidation of certain chromogens by hydrogen peroxide with the aid of a catalyst to produce a dyestuff whose presence is detected photometrically. Peroxidase and the systems, iodide/molybdate (German Auslegeschrift No. 1,284,124), iodide/vanadate (German Offenlegungsschrift No. 1,598,828) and iodide/tungstate (German Offenlegungsschrift No. 2,163,421) are known as such catalysts in connection with glucose determinations.
In another method of glucose determination (Anal. Chem. 34, 452 (1962)), the triiodide formed in the presence of the catalyst system iodide/molybdate, is directly measured photometrically without using a chromogen. This method has the advantage over the chromogen-based methods in that the high extinction coefficient of the triiodide (about 20 cm.sup.2 /.mu.M) can be utilized in the spectrophotometric determination, whereby high precision is achieved. At the same time, interference caused by concomitant colored substances in the sample is minimized. In addition, the reagent solution is very stable and can therefore be stored at room temperature, and the use of carcinogenic and cocarcinogenic chromogens can be avoided.
The decisive disadvantage of this method is that the body fluid samle to be investigated must be deproteinized beforehand because, otherwise, the coloration of the triiodide is not stable. However, the time-consuming deproteinization not only makes the determination more difficult, but in cases in which deproteinization is not possible, this requirement renders the otherwise advantageous method inapplicable. One such case is the determination of cholesterol in body fluids since the cholesterol is bound protein.